Review Article
Development 110, 1-18 (1990)
Printed in Great Britain © The Company of Biologists Limited 1990
'Generic' physical mechanisms of morphogenesis and pattern formation
STUART A. NEWMAN1 and WAYNE D. COMPER2
^Department of Cell Biology and Anatomy, New York Medical College, Valhalla, New York 10595, USA
Department of Biochemistry, Monash University, Clayton, Victoria 3168, Australia
2
Summary
The role of 'generic' physical mechanisms in morphogenesis and pattern formation of tissues is considered.
Generic mechanisms are defined as those physical processes that are broadly applicable to living and nonliving systems, such as adhesion, surface tension and
gravitational effects, viscosity, phase separation, convection and reaction-diffusion coupling. They are contrasted with 'genetic' mechanisms, a term reserved for
highly evolved, machine-like, biomolecular processes.
Generic mechanisms acting upon living tissues are
capable of giving rise to morphogenetic rearrangements
of cytoplasmic, tissue and extracellular matrix components, sometimes leading to 'microfingers', and to
chemical waves or stripes. We suggest that many mor-
phogenetic and patterning effects are the inevitable
outcome of recognized physical properties of tissues, and
that generic physical mechanisms that act on these
properties are complementary to, and interdependent
with genetic mechanisms. We also suggest that major
morphological reorganizations in phylogenetic lineages
may arise by the action of generic physical mechanisms
on developing embryos. Subsequent evolution of genetic
mechanisms could stabilize and refine developmental
outcomes originally guided by generic effects.
Introduction
diffusion (Crick, 1970) and interfacial tension (Steinberg, 1978; Heintzelman et al. 1978), participate in
important ways in morphogenesis and pattern formation. In contrast to molecular machines, which are
mainly suited to bringing about precise outcomes in
spatially localized tissue regions, some of these general
physical effects may act globally, so as to influence
tissue shape and composition over relatively long distances.
Because both highly evolved biomolecular processes
(conveniently referred to as 'genetic') and more broadly
applicable ('generic') physical processes can each contribute to any given developmental episode, investigators need to take both categories of phenomenon into
account. But research on generic morphogenetic and
patterning processes is a rapidly expanding area of
physical chemistry that is unfamiliar to most developmental biologists. This has restricted the influx of a
number of fruitful concepts into developmental biology
and impeded the use of several informative cell-free
experimental models of morphogenesis.
In what follows we will attempt to redress this
deficiency by presenting a typology of generic physical
mechanisms relevant to animal tissue behavior. These
mechanisms include familiar physical effects such as
gravity, viscous flow, phase separation and adhesion.
But they also include such exotic processes as Marangoni effects, convective fingering and chemical concentration waves. Previous applications of some of these
Developing, regenerating, healing and neoplastic tissues undergo changes in form and cellular composition
by mechanisms that are poorly understood. While all
contemporary approaches assume that tissue morphogenesis and position-dependent cell differentiation
(pattern formation) are caused ultimately by the interplay of physicochemical behaviors of macromolecules,
such behaviors fall into at least two distinguishable
categories. Certain developmental processes depend on
highly organized interactions between specific macromolecules, and can be appropriately characterized as
'molecular machines'. The existence of each such
machine presupposes the coevolution of several biological macromolecules, leading to the coordination of their
physicochemical properties in the service of a particular
function. Examples include cytoplasmic 'motors' that
affect the shape and motility of individual cells (Vale,
1987), and gene promoter elements sensitive to complexes of spatially distributed DNA-binding proteins
(Stanojevid et al 1989; Goto et al. 1989).
But there is also evidence that physical forces and
dynamical processes that are not the products of the
evolved coordination of macromolecular properties,
but are organizing principles of nonliving as well as
living systems, such as gravity (Ancel and Vintemberger, 1948; Malacinski, 1984), adhesion (Steinberg,
1978; McClay and Ettensohn, 1987; Armstrong, 1989),
Key words: pattern formation, morphogenesis, genetic
mechanism, generic physical mechanism.
S. A. Newman and W. D. Comper
mechanisms to development will be reviewed, and
additional examples will be given of developmental
processes that may profitably be analyzed in terms of
such mechanisms.
In our discussion, the morphogenetic properties of
individual cells - e.g. their extensibility, contractility
and motility - are treated as given; they are assumed to
arise from the physical chemistry of highly evolved
intracellular proteins such as tubulin, actin, myosin and
kinesin, in the presence of sources of metabolic energy
and appropriate cofactors. In this sense they are 'genetic'. Similarly, the ability of cells to undergo differentiation in response to microenvironmental signals, and
to produce and secrete specific macromolecules, is
assumed. Secreted macromolecules will be considered
here only insofar as they can potentially play the role of
dynamical components in some of the generic physical
processes that we will describe. And whereas eggs and
multicellular embryos have the ability to produce transcellular ion currents and endogenous electrical fields
that reflect morphogenetic polarity, growth and regeneration (Jaffe, 1981; Nucitelli, 1984), it is not clear that
bioelectricity as a generic phenomenon has a role in
developing systems, apart from its association with
transport and utilization of specific ions. We will therefore tentatively group these phenomena with other
active chemical processes, and refer the reader to the
reviews cited above for further details.
Our main purpose is to familiarize developmental
biologists with the range of generic physical mechanisms that can participate in biological morphogenesis
and pattern formation, and to indicate possible relationships between these processes and the highly
specific molecular interactions that also mediate developmental events and are responsible for the precision of
their outcomes. In particular, we suggest that many
morphogenetic processes may have first arisen in evolution by the action of generic physical mechanisms on
cells and tissues, and that particularly favorable results
were later stabilized and made more dependable by the
superimposition of more evolved genetically determined mechanisms. In this perspective, the de novo
origin of developmental mechanisms becomes less
problematic: contemporary molecular mechanisms
could have evolved as reinforcements for less precise
generic physical determinants, the conditions for which
may or may not currently prevail. And while the
possible generic origins of certain morphogenetic processes may be obscured by an overlay of evolved genetic
mechanisms, they can potentially be brought to light by
well-conceived experiments.
It will also be noted that many of our biological
examples relate to events involving the extracellular
matrix. This is not because generic physical processes
are prohibited from occurring intracellularly. Indeed,
cytoplasmic reorganization in the amphibian egg may
make use of such processes (see below). However, it
seems reasonable to assume that the constraints on
molecular processes within the cell are in general
greater than those in the extracellular milieu, and the
tolerance of variability correspondingly smaller. Extra-
cellular matrices, under this assumption, would have
less of the character of 'molecular machines' than most
intracellular macromolecular assemblages, and would
therefore be more typical loci for the physical processes
we have termed 'generic'.
Examples of Generic Processes
The mechanical properties of materials are conveniently described in terms of their responses to
stresses, which are forces applied to bodies of matter. A
change in the dimensions of a body produced by a stress
is called a strain. Shear stresses act tangentially to planes
within the material and cause continguous parts of the
body to slide past one another. In solids, shear stresses
are opposed by bonds between adjacent subunits and by
elastic restoring forces, whereas liquids begin to flow as
soon as a shear stress is applied. The capacity of a liquid
to flow is due to the ability of the liquid's molecules or
other subunits to readily changetheir relative positions.
The physical state of a living tissue, can span the range
from liquid (blood) to solid (bone). But it is only in
intermediate state, semisolid tissues tljat developmentally significant, short-term morphogenetic effects can
take place. Typical tissues exhibit both-elastic properties, which permit them to resume their shape when a
shear stress is removed, and viscous properties, in
which rearrangement of internal components (cells or
extracellular matrix materials) permits shape change in
response to shear stress. (Phillips and Steinberg, 1978;
Steinberg and Poole, 1982).Viscoelastic fluids can be compressible or noncompressible; that is, their volume will decrease or remain
unchanged under compressive stresses, which are forces
directed normal to planes within the material. However, tissues are generally noncompressible because of
their high water content. Even when local reductions of
extracellular space occur, as in mesenchymal tissues
undergoing condensation (Thorogood and Hinchliffe,
1975), retention of water will ensure that the overall
tissue volume is conserved.
Like other fluid systems, tissues are subject to the
ubiquitous effects of gravity and adhesion. Either of
these forces can effect shape change, but the degree of
deformation will depend on the mechanical properties
of the particular tissue (its relative elasticity and viscosity).
The nonuniform distribution within a tissue of mechanical properties, such as density or viscosity, or of
chemical components, can be sources of morphogenetic
change or cellular pattern formation. However, nonuniformities in density, viscosity or chemical composition
will tend to dissipate by mixing unless either the
different subregions are immiscible, or the nonuniformities result from an active, free energy-consuming
process that maintains the nonequilibrium heterogeneous state. The latter can only occur in systems that
are open to external sources of fuel and raw materials,
conditions well-satisified by living tissues. The possibility of maintenance of spatially nonuniform chemical
Physical mechanisms of morphogenesis and pattern formation
composition in a tissue by the coupling of biosynthesis
and diffusion (Turing, 1952; Prigogine and Nicolis,
1967; Nicolis and Prigogine, 1977) is discussed in a later
section.
The formation of immiscible phases can preserve
spatial heterogeneity even without the expenditure of
free energy. Phases are physically distinct, mechanically
separable portions of matter in a single heterogenous
physical system. Oil and water are a familiar example of
distinct fluid phases, but cytoplasm and yolk in amphibian eggs interact in a similar fashion. In fact, as we will
describe below, tissues from different sources often
behave as distinct fluid phases. Their immiscibility can
result from differences in strengths of homotypic and
heterotypic cell interactions (Steinberg, 1978), or by
virtue of organizational properties of their extracellular
matrices (Forgacs et al. 1989) (see below).
Immiscible fluid phases are characterized by a tension
at their common interface. This interfadal tension
represents the incremental increase in free energy
brought about by attempting to deform the interface at
constant temperature, pressure and composition. A
zero interfacial tension is tantamount to miscibility of
the subregions. The relative balance of the cohesive
interactions within each phase and the adhesive forces
between them defines the interfacial tension, as well as
determining the contour and extent of the boundary
between the regions. Quantitative modulation of the
strength of adhesion at the boundary between two
liquid phases, and at the boundary of each phase with
any tissue surface with which it comes into contact, can
induce subtle or gross morphogenetic rearrangements.
The relevance of these 'wetting' (de Gennes, 1985) or
spreading effects to the behavior of living tissues will be
discussed below.
Morphogenetic changes driven by local disparities in
interfacial tension are well-known in nonliving systems.
Droplets of fluid along the interface are driven in the
direction of increasing tension, and the resulting shear
stresses induce motion in the bulk of the interfadng
fluids. An example is the 'tears of strong wine', in which
finger-like streamers form at the air-liquid interface in
a wine glass. These phenomena are collectively referred
to as Marangoni effects (Scriven and Sternling, 1960;
Napolitano, 1984), and may also be relevant to tissue
morphogenesis.
Differences in density, viscosity or composition between subregions in a heterogenous particle suspension
or multicomponent fluid (and, by implication, a tissue
or egg), even in the absence of interfacial tension
between the regions, when subject to force fields like
gravity or pressure, may give rise to a variety of bulk
flow patterns. These range from simple attainment of
gravitational equilibrium by 'preloaded' components
that have been released from mechanical constraint, to
more unusual effects such as interdigitated convective
'microfingers' (Saffman and Taylor, 1958; Preston et al.
1980; Davis and Acrivos, 1985; Nittmann and Stanley,
1986). Like the other generic processes outlined above,
convective fingering effects may be embodied in morphogenetic and patterning mechanisms.
Finally, we note that many of the generic processes
considered below exhibit nonlinear responses to relevant control variables. That is, small changes in
interfacial tension, density, biosynthetic or diffusion
rates, or even size and shape of a tissue domain, can
lead to profound changes in the resulting morphology.
This clearly bears on the succession of pattern and form
during development. As we shall see, it also has
implications for the evolution of phylogenetic lineages.
In what follows, we will attempt to situate generic
mechanisms within the framework of existing developmental studies. The major generic effects considered in
this review, their driving forces and their outcomes, are
summarized in Table 1.
Phase separation and vlscoelastic behaviors of
embryonic tissues
Steinberg and his coworkers (Steinberg, 1962; Phillips
and Steinberg, 1978; Steinberg and Poole, 1982) have
considered the morphogenetic behavior of tissues in
terms of their resemblance to liquids. Tissue fragments
can flow in response to external forces, round up when
suspended in a fluid medium, and coalesce with other
such fragments, much like liquid droplets. Mixtures of
cells from different types of tissue will sort out into
homotypic 'islands' and 'lakes', and will eventually
separate out completely, like a suspension of oil in
water (Steinberg and Poole, 1982). After sorting out has
occurred the relative configurations of the tissues are
what would be predicted if tissues, like simple liquids,
exhibited interfacial tensions with respect to their
surroundings. In the case of tissues these surroundings
could be other tissues, culture media, or artifidal
substrata (Fig. 1).
In terms of the categories and definitions of the
previous sections, Steinberg's analysis asserts that many
morphogenetic movements of tissues are explicable on
the basis of the progression to equilibrium of closed,
multiphase liquid systems. However, it is clear that
living systems, which have both nutritional and waste
removal requirements, cannot be truly closed, and that
different tissue types are not uniform physical phases in
the sense of the definitions above. Furthermore, cells
and the tissues they constitute can actively generate
motile and contractile forces, unlike liquids which flow
passively. Finally, the molecular complexity of adhesive
interactions between similar and dissimilar cell types
(McClay and Ettensohn, 1987) seems difficult to reconcile with the explanation of equilibrium tissue configurations on the basis of a single quantitative scale of
surface tension. In what sense, therefore, is there
validity to Steinberg's application of the generic physical mechanism of equilibrium phase separation of
immisdble liquids to tissue morphogenesis?
First, the liquid nature of certain tissues is an inevitable consequence of the fact that cells can slip past one
another (Phillips et al. 1977) and dissipate strains that
would otherwise cause bending or warping, changes in
form that are characteristic of solids. The slippage may
S. A. Newman and W. D. Comper
Table 1. Generic morphogenetic and patterning mechanisms considered in this review
Mechanism
Phase separation;
wetting
Driving force
Tissue feature acted upon
Outcome
References
interfacial tension
adhesive differences;
viscosity
tissue spreading
& engulfment;
cell sorting out
elasticity
folding &
distension of
epithelial sheets
Torza & Mason (1969)1;
Newman et al. (1985)';
Forgacs et al. (1989)';
Steinberg (1978)2;
Armstrong (1989)2;
Frenz et al. (1989a,b)2
Mittenthal & Mazo (1983)1'2
Marangoni effect
interfacial tension
local variations in
adhesion along
common interface
interdigitating
microfingers
Scriven & Sternling (I960)1;
Napolitano (1984)1;
Conklin (1905)2;
Horstadius & Sellman (1946)2
Sedimentation
and buoyancy
gravity
density differences
ooplasmic segregation
& rearrangement
Ancel & Vintemberger (1948)2;
Malacinski (1984)2;
Neff et al. (1984)2
Convective flows
gravity
local variations in
density
interdigitating
microfingers
Preston et al. (1980)1;
Comper et al. (19870,6)';
Conklin (1905)2;
Horstadius & SeUman (1946)2
Reaction—diffusion
coupling
chemical potential,
positive feedback
biosynthesis; tissue
permeability
chemical waves
Turing (1952)1'2;
Gierer (1981)1'2;
Meinhardt (1982)1'2;
Newman & Frisch (1979)1-2;
Newman et al. (1988)1'2;
Meinhardt (1988)1-2
1
2
Physical mechanisms.
Possible biological roles.
be purely passive, as with the molecular subunits of a
non-living liquid, or can be facilitated by random
motility of the living cells. Because of such internal
rearrangements many tissues will not bend under application of force, or bounce back when it is removed: they
will flow instead.
Second, subunits (molecules or cells) of a given liquid
have characteristic binding interactions with one
another despite their relative mobility. While there is
no a priori physical reason why subunits in an arbitrary
liquid should adhere to subunits of the same kind in
preference to subunits from another liquid, it is a wellrecognized fact of tissue biology that cells generally
adhere to their own type in preference to other cell
types (Townes and Holtfreter, 1955). This feature
places tissues in a restricted category of liquids: they
will generally be immiscible with one another. That is,
when intermixed, tissues or their constituent cells will
separate into distinct phases, and when confronted with
one another, they will undergo characteristic spreading
behaviors, not simply in analogy to oil and water, but
for the same thermodynamic reasons. Inert particles
can also be transported through tissues (Wiseman,
1977; Frenz etal. 1989a), most likely by adhesion-driven
processes similar to those responsible for heterotypic
cell sorting. Such particles can be used as probes of the
adhesive environment in morphogenetically active tissues.
It should be noted that the arguments presented
above hold whether homotypic adhesive preference of
cells is achieved by quantitative differences in the
strength of a single molecular adhesive mechanism, or
by the more biologically plausible use of qualitatively
different adhesion molecules by the two cell types. This
is precisely because, in considering interactions between different types of cells, adhesive preferences
based on qualitative differences in binding mechanisms
can be ordered uniquely on a scale of greater/lesser
binding strength as if the differences were simply
quantitative.
Any two immiscible liquids will exhibit a characteristic interfacial tension when brought into proximity.
Because a change in the area of the interface has a cost
in terms of free energy, for a system at equilibrium,
where the total free energy is at a minimum value
consistent with all internal and external constraints, the
interface will have a well-defined shape. In other words,
the degree of mutual spreading of contiguous liquids, or
engulfment of one liquid (or tissue) by another, will
depend on the relative strength of the interfacial
tension between the two liquids compared with the
tensions at the interfaces of the two liquids with their
other bounding substrata (Torza and Mason, 1969)
(Fig. 1A,B). Because the relative balance of adhesive
interactions at the various interfaces determines the
extent of spreading, adhesion or deadhesion of a cell
Physical mechanisms of morphogenesis and pattern formation
population to different bounding surfaces can drive its
concerted translocation in new directions (Forgacs et al.
1989).
Individual cells also exhibit fluid properties, and can
locomote along adhesive substrata by an interfacial
tension-driven process. This phenomenon has been
termed 'haptotaxis' by Carter (1967). However, a single
cell can only progress along its substratum by haptotaxis
insofar as the substratum becomes progressively more
adhesive over distance, i.e. if there is an .adhesive
gradient. In contrast, the concerted movement of populations of cells can be accounted for by the liquid-like
spreading of the tissue as a whole along a substratum.
This substratum must be adhesive, but need not contain
an adhesive gradient. The apparent dependence of
unidirectional migration of neural crest cells on intercellular contact (Newgreen et al. 1982) has been interpreted in terms of contact inhibition of movement
(Newgreen and Erickson, 1986). But it may equally well
reflect the propensity of such cells to interact with their
adhesive environments as parts of coherent fluid tissues.
The spreading of the chick blastoderm on the inner
surface of the vitelhne membrane (Downie, 1976), the
B
1A 7
node
C ectoderm
ierm
p n r l n r l p r m
•:'::
••:..••.•
:.V.-.-.:
,
._
mesoderm
(still invaginating)
Fig. 1. Interfacial tension-driven effects in physical systems and possible roles in tissue interactions. (A) Photographs of
stages of complete engulfing of water containing 1 percent malachite green (black drop) by a drop of polyglycol oil. Both
drops were suspended in silicone oil. Frames 1 to 6 show the penetration of the aqueous phase into the oil phase, and
frames 7 to 9 show the subsequent relaxation of the deformed water drop into the oil drop. Total elapsed time was 0.9s
From Torza and Mason (1969). Copyright 1969 by the AAAS. Picture courtesy of Dr T. G. M. van de Ven. (B) Spreading
of 10-day-old chick embryo pigmented retinal tissue over the surface of an aggregate of 10-day-old embryonic heart tissue.
Approximately spherical aggregates of the two tissues were placed in contact in hanging drop culture until they were firmly
in adhesion, after which the composite aggregate was maintained in organ culture for 2 days. During this time, the
pigmented retinal tissue spread as a monolayer over the surface of the heart aggregate. From Armstrong (1989). Picture
courtesy of Dr Peter B. Armstrong. (C) Stages in mammalian gastrulation viewed in median section. (Left) Axial mesoderm
has begun entering the primitive pit, forming the notochordal process or chorda. (Right) Chorda advances beneath the
ectoderm followed by additional mesoderm, which then spreads laterally (out of the plane of section). Redrawn from
Deuchar, 1975. (D) Matrix-driven translocation experiment (Newman et al. 1985) performed between two polystyrene plates
(Forgacs et al. 1989), viewed from the side. Primary gel consists of cells or polystyrene beads suspended in type I collagen.
Secondary gel contains 12.5/ig ml"1 fibronectin in type I collagen. Spreading of primary gel along interfaces between
secondary gel and upper and lower substrata occurs over approximately 5min.
5. A. Newman and W. D. Comper
spreading of the chick embryonic epicardium over the
myocardium (Ho and Shimada, 1978), gastrulation
(Phillips and Davis, 1978) (Fig. 1C), teleost epiboly
(Armstrong and Child, 1965), the elongation of the
salamander pronephric duct over the lateral mesoderm
(Poole and Steinberg, 1981), and the condensation of
precartilage mesenchymal cells (Frenz et al. 1989/?) are
only some of the apparently goal-directed processes in
the embryo likely to be driven, at least in part, by
interfacial forces acting on liquid tissues.
Of course, tissues will not exhibit purely viscous
behavior. The potential development of nonuniform
tensions within connective tissues as a result of cell
action on surrounding matrix fibers is a feature attributable to their partly elastic nature. The morphogenetic
consequences of this property have been studied in
model tissues consisting of cells suspended in defined
collagen matrices (Bell et al 1979; Harris et al. 1981).
The formation of 'compartments' in the developing
insect cuticle (Crick and Lawrence, 1975; Garcia-Bellido et al. 1976) can be plausibly treated as the progressive establishment of immiscible domains in epithelial
sheets. Epithelia are interesting materials in that their
cells are capable of exchanging neighbors (Keller,
1978), making them fluid in a two-dimensional plane.
With respect to motions out of the plane, however,
epithelia can act as elastic sheets. Indeed major features
of epithelial morphogenesis can be explained in terms
of deformations of epithelial sheets arising from strains
generated by interfacial tensions between adjacent
tissue blocks (Mittenthal and Mazo, 1983).
Mesenchymal and epithelial tissue behaviors thought
to be due to generic physical mechanisms are inevitable
consequences of recognized properties of these tissues
such as homotypic adhesive preference of cells, and
viscosity and/or elasticity. It is reasonable to suppose
that genetically specified molecular mechanisms have
evolved to reinforce these inherent tendencies, and to
limit or specify the conditions for their occurrence. The
evolution of mechanisms indifferent to, or in opposition
to these forces, while formally possible, would probably
have occurred less frequently.
Phase separation and adhesion-promoted cell
transport in model connective tissues: matrixdriven translocation
The potential contribution of interfacial tension-driven
effects to tissue morphogenesis can be demonstrated
persuasively in a cell-free extracellular matrix system in
which cytoplasm-generated motile forces play no role.
If cells or cell-sized polystyrene latex beads with
heparin-like molecules on their surfaces are suspended
in a solution of type I collagen, and a drop of this
suspension is placed contiguous to a second collagen
drop, a sharp interface can form between the two
regions. For this to occur, both the collagen and the
particles must be in a certain concentration range
(Newman et al. 1985; Forgacs et al. 1989). If the
adhesive glycoprotein fibronectin is present in the
second droplet there is a concerted translocation of
particles and surrounding matrix, resulting from the
expansion of the interface between the droplets near
both bounding surfaces (Fig. ID; Fig. 2). This effect,
termed 'matrix-driven translocation' highlights certain
generic physical properties of extracellular matrices
that could readily play a part in morphogenetic processes in living tissues.
One of these properties is the apparent 'phase
constituting' effect of extracellular fibers (Forgacs et al.
1989). The relative movement of matrix subregions by
matrix-driven translocation could not occur if the regions remained miscible; a true interface is required
(Forgacs et al. 1989). Surprisingly, under the minimal
conditions required for translocation, the cells or beads
in the initially populated region are, on the average,
located more than ten particle diameters apart. Previous accounts of tissue engulfment behavior attributed
the immiscibility of tissues of different origin to the
adhesive preference of at least one of the interacting
cell types for cells of its own kind (Steinberg, 1978;
Steinberg and Poole, 1982). In the matrix-driven translocation system (and in the mesenchymal tissues for
which it serves as a physical model), a mechanism must
exist to promote phase formation where cells are not
directly in contact.
'Percolation theory' (Stauffer, 1985) is a mathematical tool for analyzing cluster formation in physical
systems as varied as the gelling of polymers (Bouchaud
et al. 1986), formation of galaxies (Schulman and
Seiden, 1986) and the spreading of forest fires (MacKay
and Jan, 1984). Using this approach it has been shown
that the relative concentrations of cells and extracellular fibers in the matrix-driven translocation system, and
in typical mesenchymal tissues, are sufficient to induce
'macroscopic clusters', or networks, in subregions of
tissues. According to percolation theory, the formation
of such a network causes phase separation of the region
containing it from similar regions that do not. The
resulting interface provides the conditions for coherent
movement of populations of cells by interfacial tensiondriven effects (Forgacs et al. 1989). These studies also
suggest that tissue regions populated with cells and
adjacent nonpopulated extracellular matrices can constitute distinct physical phases, afindingthat is relevant
to the analysis of gastrulation, neural crest migration,
and other processes in which cell populations enter cellfree spaces (See Trelstad (1984) for reviews).
Another property of the matrix-driven translocation
system that bears on mechanisms of morphogenesis in
living systems is the demonstration that the spontaneous spreading behavior of tissues may be impeded
by low energy hindrances at their interfaces, which can,
in turn, be overcome by specific, but energetically
weak, molecular interactions. The force for the concerted movement of cell or bead suspensions in the
matrix-driven translocation system is related to the
reduction in free energy brought about by spreading of
one collagen phase upon the other. This spreading can
occur in the absence of fibronectin at collagen concentrations slightly below that required for the fibronectin-
Physical mechanisms of morphogenesis and pattern formation
±FN
TOO FEW BEADS
NO TRANSLOCATION
NO PHASE SEPARATION
TOO FEW LINKERS
NO TRANSLOCATION
NO PHASE SEPARATION
CRITICAL NUMBER OF
BEADS AND LINKERS
INTERFACE FORMS
TRANSLOCATION
PHASE SEPARATION
BOUND INTERFACE
NO TRANSLOCATION
EXCESS OF LINKERS
PHASE SEPARATION
UNBOUND INTERFACE
TRANSLOCATION
dependent effect (Forgacs et al. 1989; Fig. 2). At the
higher collagen concentration this spontaneous spreading is hindered (possibly by excess binding of particles
to matrix at the interface), but can be released by the
weak, but highly specific interaction of the cell or bead
surfaces with the amino-terminal heparin-binding
domain of fibronectin (Newman et al. 1987; Khan et al.
1988; 1990).
In complex cell-matrix systems, where there are
multiple adhesive interactions, the balance of forces can
potentially be tipped by weak interactions. This provides a likely locus for the intersection of generic and
genetic mechanisms. In the model system described, for
example, the generic phenomenon of the mutual
spreading of two liquids provides the driving force for a
morphogenetic event that can be hindered or released
Fig. 2. Interpretation of
formation of separate tissue
phases by generation of networks.
Cells or polystyrene particles with
appropriate surface characteristics
('beads') are mixed with soluble
collagen and placed next to a drop
of collagen which contains no
particles, but may or may not
contain a substance adhesive to
the cell or bead surface
(fibronectin: 'FN'). When too few
particles or too few collagen fibers
('linkers') are present, no
pervasive network forms, and no
phase separation takes place.
When a critical number of
particles and linkers are present
network formation and phase
separation occurs, and one phase
may tend to spread upon or engulf
the other. At higher collagen
concentrations spreading may be
impeded and require adhesive
interactions at the interface to
overcome the hindrance. From
Forgacs et al. (1989).
by weak but highly specific biomolecular interactions at
the interface.
Gravity and cytoplasmic reorganization In the
amphibian egg
Another illuminating example of the potential relation> ship between generic and genetic mechanisms in morphogenesis is provided by the amphibian egg. Eggs of
anuran species have a thin, outer cortical layer of
cytoplasm that is immiscible with the central egg
cytoplasm. Following fertilization in anurans there is a
30° rotation of this cytoplasm relative to the deeper
cytoplasm (Ancel and Vintemberger, 1948; Vincent et
al. 1986; Fig. 3). In some species this rotation has the
8
S. A. Newman and W. D. Comper
SEP.
SEP
Fig. 3. The cortical/cytoplasmic rotation in anuran
embryos. A 30° rotation of the cortex relative to the inner
cytoplasm is required for normal dorsoventral polarity to be
established. Diagrammatic sections are shown before (left)
and after (right) rotation. The cortex rotates so that the
sperm entry point (SEP) moves vegetally. The grey crescent
visible in Rana pipiens embryos is formed by the
overlapping of pigmented animal pole cytoplasm by
nonpigmented vegetal pole cytoplasm. Redrawn from
Elinson and Rowning (1988).
effect of revealing a lightly pigmented region of deep
cytoplasm on one side of the egg, the grey crescent.
This region, or its equivalent, marks the future dorsal
area of the embryo.
Inhibition of the cortical cytoplasmic rotation by UV,
cold shock, or chemical means severely perturbs dorsoventral axis formation in the embryo. In partially
inhibited eggs, the amount of rotation is correlated with
the extent of axis formation (Vincent and Gerhart,
1987). Moreover, in inverted eggs, the direction of shift
of major cytoplasmic components determines the dorsoventral polarity of the ensuing embryo (Gerhart et al.
1981; Neff et al. 1984). These experiments have provided persuasive evidence that subcortical rotation is a
necessary early step in embryonic axis specification.
Ancel and Vintemberger (1948) suggested that the
relatively rigid egg cortex, upon mechanical release
from the underlying cytoplasmic core midway during
the first cell cycle, slips to one side by 30° under the
influence of gravity, in normally oriented eggs. The
twinning effects of unit gravity on experimentally
rotated eggs (Gerhart et al. 1981), and the ability of unit
gravity to effect a proper overlap between cortical and
deep cytoplasm in eggs whose normal rotation is
inhibited by UV (Neff et al. 1984; Vincent & Gerhart,
1987), are consistent with a role for gravity in normal
axis specification.
However, a mechanism of axis specification driven
exclusively by gravity is contradicted by results of
Vincent et al. (1986), who embedded Xenopus eggs in
gelatin so that the cortex could not move, and found
that the cytoplasmic core, including the denser vegetal
regions, rotated up one side of the cortex by 30°,
working against gravity. Clearly the egg must also have
a means other than gravity to drive the rotation.
The presence of an oriented array of microtubules in
the shear plane between the cortex and subcortical
cytoplasm (Elinson and Rowning, 1988) suggests that
the force-generating mechanism might be similar to the
energy-consuming microtubule-dependent organelle
transport systems found in other cell types (Vale, 1987).
But because gravity may suffice to drive cytoplasmic
reorganization under all but the most unusual circumstances, it could have been the phylogenetically original
determinant of cortical rotation and axis specification.
A specific microtubule-based force-generating mechanism may have subsequently been selected on the basis of
its ability to enhance the dependability of an event
originally driven by a generic physical process.
Convectlve mechanisms
Concerted relative motion of different regions of a fluid
is known as convection (Velarde and Normand, 1980).
If the different fluid regions have an interfacial tension
between them, convective flows can take place by the
progression to adhesive equilibrium from a metastable
state, as proposed for the matrix-driven translocation
system above. Flows can also result from instabilities in
the contour of the interface due to local variations in
interfacial tension. These variations can be brought
about, in turn, by local changes in composition along
the interface. Flows resulting from variations in the
interfacial tension are examples of Marangoni convection (Scriven and Sternhng, 1960). Both classes of
phenomena depend on generic mechanisms that can
occur in living tissues.
Even in the absence of interfacial tension, different
parcels of a fluid may undergo convection if they differ
in density or viscosity, and a force that can act on these
differences is present. Regions of different density of a
heterogeneous cytoplasm, tissue or extracellular
matrix, if they are out of gravitational equilibrium, are
clearly subject to morphogenetic rearrangement by
gravity as indicated in the previous section. Contiguous
cytoplasmic compartments, tissues or matrices that
differ in viscosity (a measure of internal frictional forces
relative to flow velocity) will flow relative to one
another if subjected to external pressure.
Convection in physical systems is commonly brought
about by temperature gradients, which would not
typically occur in developing tissues. But the proximate
causes of thermal convection, as in some of the
examples discussed above, are actually density or surface tension inhomogeneities, which, at least in tissues,
can result from metabolic or biosynthetic processes
rather than heating.
A number of developing systems exhibit a microfinger morphology in which distinct cytoplasmic or
tissue components interdigitate with one another. The
width of each microfinger is usually on the order of tens
to hundreds of pcm. Some examples are shown in Fig. 4.
These microfingers can be transient, like the residual
streamers resulting from downward flow of yellow
cytoplasm in the newly fertilized egg of the ascidian
Styela partita (Conklin, 1905) (Fig. 4A), or in the
migration pattern of endogenous and ectopic neural
crest cells in the axolotl embryo (Horstadius and
Sellman, 1946) (Fig. 4B). They can also be stable, as in
the fully developed pattern of kidney collecting tubules
Physical mechanisms of morphogenesis and pattern formation
yellow corticol cytoplasm
gray yolk
yellow cytoplasi
(Fig. 4C). These microfingers suggest the action of
convective mechanisms, which, in multicomponent
fluids with or without interfacial tension (Saffman and
Taylor, 1958; Nittmann and Stanley, 1986) or in settling
particle suspensions (Davis and Acrivos, 1985), can give
rise to microfingers on the scale seen in these living
systems.
Suspensions of cells or inert particles can spontaneously organize into countercurrent microfinger patterns under the influence of gravity. Countercurrent
streaming associated with random density fluctuations
has been observed in Tetrahymena cultures (Winet and
Jahn, 1972). Microfinger morphologies have also been
observed during sedimentation of polydisperse and
binary particle suspensions at high solid concentration
(reviewed in Davis and Acrivos, 1985). Whitmore
(1955) performed experiments with suspensions containing a mixture of heavy particles and neutrally
buoyant particles. The initially homogeneous suspension quickly organized into a stratified system with
microfingers moving in the vertical plane: the buoyant
particles (~100/xm in diameter) were carried in the
upward moving fingers and the heavier particles in the
downward moving fingers. More recently, Weiland and
McPherson (1979) found that after adding buoyant
Fig. 4. Microfinger patterns in living and nonliving systems.
(A) Cytoplasmic rearrangement in the egg of the tunicate
Styela partita. (Left) Before fertilization, inner gray yolky
cytoplasm is surrounded by a peripheral layer of yellow
cytoplasm. (Right) By five minutes after fertilization the
yellow cytoplasm has streamed to the vegetal pole, exposing
the gray yolk. Microfingers of yellow cytoplasm continue to
flow vegetally. Redrawn from Conklin (1905). (B) Normal
and ectopic cranial neural crest migration in the axolotl
embryo. (Left) The right neural ridge of the head has been
stained with neutral red (coarse stippling). The left ridge has
been excised, stained with Nile blue and implanted
horizontally lower down on the same side (fine stippling).
(Right) Red-stained ectomesoderm from the right side is
migrating down on the left side, as it does normally, meeting
streams of cells that migrate from the graft in the dorsal
direction. Redrawn from HSrstadius and Sellman, 1946.
(C) Autoradiographic image of a slice of rat kidney perfused
with a tritiated gaseous compound. The filled collecting
ducts, which are between
30pan and 50/an wide, are
evident in a microfinger
arrangement. From Charpak et
al. (1989). Picture courtesy of
Dr G. Charpak. (D) Time
evolution of structured flows in
a polymer system containing
dextran and poly(vinylpyrrolidone) (PVP). The PVP
in the lower layer was coupled
to a blue dye. (Left) Initial
preparation; (Right) After
40min. The microfingers are
on the order of 500 /an in
width. Adapted, with changes,
from Comper et al. (1987a).
particles to an otherwise uniformly settling suspension,
there was a rapid lateral segregation of the two species
of particles into countercurrent vertical fingers. The
mechanisms for these gravity-driven convective patterns involving large particles is not known, but clearly
they may be relevant to living systems with a heteropycnic population of cells that can be made to flow relative
to one another.
Remarkably, gravity-driven convective microfingers
can even occur in spatially nonuniform multicomponent
fluid systems that are initially in gravitational equilibrium, i.e. where the denser fluid is layered below the
less dense fluid. This latter effect is a general property
of polymer solutions, and has been demonstrated for
proteoglycans (Harper et al. 1984), collagen undergoing
fibrillogenesis (Ghosh and Comper, 1988), and substrate and product gradients in the presence of a
converting enzyme (Comper and Preston, 1981), and
predicted to occur for the assembly-disassembly of
microtubules in axons (Comper et al. 1983). The origin
of the effect resides in the fact that relaxation of
concentration gradients in multicomponent systems is
often accompanied by the formation of 'microdensity
inversion' (Comper et al. 1987/)). This is an unstable
state in which a layer of denser fluid transiently overlays
10
S. A. Newman and W. D. Comper
a thin layer of less dense fluid. The heavy layer tends to
sink and the lighter layer to float in striated countercurrent flows. This dynamic stratification is maintained by
a constant exchange of material between flows, and by
gravity acting on density gradients at the interface of
oppositely moving flows. In an unrestrained system
these flows grow in the vertical plane (Fig. 4D).
Convective microfingers or flows can passively transport inert particles or living cells at a rapid rate (i.e.
with velocities of mm per day to mm per min). Such
flows are able to move through 90° and 180° bends in
capillary tubing. In these cases, the upward moving
flow remains uppermost to the surface of the tubing
with respect to the downward moving flow. The flows
may rotate with respect to one another in attaining
these positions.
Whereas gravity provides the driving force in the
experimental systems described, it causes transport in a
direction counter to what would be expected on the
basis of initial density distribution. Moreover, it can
exert its effect with only a minimal gravitational gradient. Indeed, microfinger formation and rapid transport
of cells can occur in systems displaced as little as 4° from
the horizontal (Comper et al. 1987a). This makes a
morphogenetic role for these processes reasonable,
since the probabihty is very low that any channel or
mechanically constrained space in a developing organism would remain perfectly horizontal. In addition, the
dependence of the rate of flow on the magnitude of the
gravitational field is very weak: the measured rate of
transport in one such system was proportional to g° 2
(Preston et al. 1983); a flow rate proportional to g"33
has been predicted on theoretical grounds for a general
class of convective systems (Turner, 1973). Consequently, experimentally neutralizing or attenuating the
effect of gravity-driven flows by changing the orientation of the system may be difficult because of the low
exponent of gravity. One might falsely conclude that
gravity is not essential to transport if the rate of flow
were insensitive to these manipulations.'
The possibility that gravity-driven convection can
occur in the extracellular matrices of developing embryos and provide guidance for cell translocation has
been considered (Comper et al. 1987a). This may be
relevant to the microfinger morphology described for
neural crest migration by Horstadius and Sellman
(1946) (Fig. 4B), although the possibility that the invading sheet of neural crest cells is divided into streams by
anatomical obstructions (Noden, 1988) must certainly
also be considered. It is interesting, however, that
ventrally transplanted ectopic neural crest cells also
break into microfingers of similar dimension in a region
of the embryo distant from the endogeneous streams
(Horstadius and Sellman, 1946; Fig. 4B). This parallels
the countercurrent aspects of the convective processes.
The possibility of convective flows in the extracellular
matrix may also be relevant to studies by BronnerFraser (1982), who demonstrated that nonmotile cells
or latex beads were translocated along neural crest
pathways into which they had been implanted. The
translocation of inert particles was highly selective with
respect to particle surface characteristics in BronnerFraser's experiments, a property not exhibited by
matrix flows driven purely by gravity. However, gravity-driven convection may act in a cooperative fashion
with matrix-driven translocation (see previous section).
In this case, interfacial interactions between oppositely
moving flows would ensure that cells or particles with
different surface characteristics are translocated selectively.
Undoubtedly cells also use their ability to locomote
independently of one another in translocating through
the embryo (Newgreen and Erickson, 1986). But the
possible contribution to cell translocation of generic
effects such as gravity-driven and interfacially-driven
convection within the extracellular matrix may help
explain the microfinger morphology of the collective
movement of these cells, as well as aspects of the
movement that occur independently of intrinsic cell
motility.
Reaction-diffusion mechanisms
Diffusible substances can elicit various responses from
cells, and can thereby act as determinants of morphogenesis or pattern formation ('morphogens'). Without
active processes to maintain them, gradients* of diffusible substances will dissipate over relatively short time
intervals in regions of the size of embryonic fields;
therefore sources and sinks of the relevant agents or
their precursors or metabolites are minimally required
for spatial patterns that are stationary (i.e. unchanging
with time). In contrast to convection, which leads to
change in the relative positions of physically distinct
cytoplasmic, tissue or extracellular matrix components,
chemical non-uniformity does not by itself constitute a
morphogenetic outcome. Generic processes that can
reasonably lead to chemical gradients in tissues are
described below, but the morphogenetic and patterning
roles of these gradients are only manifested when cells
respond to local concentrations of the relevant substances by undergoing mechanochemical or differentiative changes that are, by and large, 'genetic'
Localized or monotonically distributed sources or
sinks of diffusible morphogens will readily lead to
stationary monotonic gradients across a tissue domain
or field (Crick, 1970). Less obvious was the discovery by
Turing (1952) that feedback interactions between reacting and diffusing components could lead to complex
stationary gradients of morphogens that could mimic,
and perhaps provide the basis for, periodic embryonic
structures.
These patterns depend on the consumption of free
energy to maintain themselves as spatially nonuniform,
but temporally constant, entities. Along with stable
convective patterns (which also can be maintained by
•In common biological usage 'gradient' is taken to mean a
monotonic distribution of a substance. Here we use the term
in the more inclusive physical sense of any nonuniform
distribution, regardless of shape.
Physical mechanisms of morphogenesis and pattern formation
the expenditure of energy), stationary chemical nonuniformities produced by reaction-diffusion coupling in
open systems have been generalized into a theory of
'dissipative structures' by Prigogine, Nicolis and their
coworkers (Prigogine and Nicolis, 1967; Nicolis and
Prigogine, 1977; Prigogine, 1980). The mechanism for
establishment of these unusual states in reactiondiffusion systems can be understood by a graphical
analysis first presented by Maynard Smith (1968)
(Fig. 5A-E).
We will make the following assumptions:
(i) two substances, A and B, which influence one
another's synthesis and consumption, are produced
throughout a row of cells.
(ii) there is a balance of the rates of transport of
precursors and metabolites of A and B, and the rates of
synthesis and consumption of A and B, such that both
substances are at steady-state concentrations within the
row of cells.
For most biochemically feasible interactions between
A and B, the steady-state concentrations of these
molecules will not vary along the row of cells, even if
one or both of them can diffuse (Fig. 5A). However,
under special circumstances a different steady-state can
be attained, in which the concentrations of A and B
change from point to point within the tissue. To see how
this may occur, consider some additional assumptions:
(iii) A has a positive effect on the synthesis of both A
andB
(iv) B has an inhibitory effect on the synthesis of A
(v) B diffuses faster than A
The consequence of these assumptions is that if the
concentration of A undergoes a small local fluctuation
to a value above its uniform steady-state level (Fig. 5B)
it will cause additional A and B to be formed (Fig. 5C).
Because B diffuses out faster than A, at some distance
away from the initial peak B will be inhibiting the
synthesis of A in cells in which A is at its original
uniform steady-state level (arrow, Fig. 5C). At this
point A will decline below that concentration (Fig. 5D),
causing the concentration of B also to drop (Fig. 5E).
It can be seen that this process will lead to a series of
peaks and valleys in the concentrations of both A and
B. In some systems a nonuniform distribution of
morphogens will be attained in which production and
breakdown of each component will balance at every
point along the tissue, so that the final distribution,
although spatially nonuniform, will be unchanging with
time, i.e. it will constitute a new steady-state. The
number of peaks and valleys that will be in place when
the system finally reaches this steady-state will depend
on reaction and diffusion rates, the size and shape of the
spatial domain in which these events are occurring, and
the modes of utilization of A and B at the boundaries of
the domain.
The example analyzed above is only one of several
generic reaction-diffusion mechanisms that can give
rise to periodic patterns (Turing, 1952; Prigogine and
Nicolis, 1967; Nicolis and Prigogine, 1977). A feature
common to virtually all of them is the presence of a selfenhancing or 'autocatalytic' property of one of the
11
components, and inhibition of the less mobile component by the more mobile one (Gierer, 1981; Meinhardt, 1982). Although reaction-diffusion processes
demonstrably lead to the formation of intricate,
spatially nonuniform patterns in nonliving chemical
systems (Ross etal. 1988; Castets etal. 1990), biosynthetic pathways are typically self-limiting rather than selfenhancing, a fact that has restricted the serious consideration of such processes as feasible developmental
mechanisms. This reservation has now been removed
with the discovery of self-enhancement of the biosynthesis of certain soluble growth and differentiation
factors, such as transforming growth factor beta (TGFP) (Van Obberghen- Schilling et al. 1988), and of some
gene regulatory proteins, such as c-jun (Angel et al.
1988).
In developing systems self-enhancement and crossinhibition of key factors may be direct, but they may
also be indirect. Despite this potential complexity,
testable hypotheses can be framed for biological pattern
formation based on reaction-diffusion processes. This
is because many predictable properties of such systems,
such as symmetries of allowable patterns, dependence
of pattern on reaction and diffusion rates of key
components, and changes in steady-state patterns
resulting from changes in tissue size and shape, can be
relatively robust with respect to variations in underlying
biochemistry (Newman and Frisch, 1979; Newman et al.
1988).
Some periodic patterns that form during embryogenesis of insects and vertebrates, and which have been
suggested to be due, in part, to reaction-diffusion
patterning mechanisms, are shown in Fig. 5F and G.
The striped pattern of expression in Drosophila embryos of the primary pair-rule genes (the initial pattern
of the even-skipped (eve) protein product is shown)
depends on the prior nonuniform distribution of gap
gene products, none of which exhibits spatial periodicity at this stage (Jackie etal. 1986). Meinhardt (1988)
has presented a model for the patterned expression of
the pair-rule genes, which assumes that these genes
specify components of reaction-diffusion pattern-forming systems. A pair-rule gene is presumed to be
activated at the borders of regions of expression of a
gap gene; autocatalysis and lateral inhibition in the
hypothesized pair-rule system ensures that stripes will
form, but only if the autocatalysis saturates at low
concentrations of pair-rule gene product. The formation of patches, rather than stripes, of pair-rule product
is sometimes seen in embryos carrying a mutation of a
pair-rule gene. This morphology is predicted from
purely dynamical considerations when saturation of
autocatalysis is not achieved (Meinhardt, 1988).
In contrast to this proposed account of the striped
pattern of pair-rule gene expression are findings by
several groups that these genes contain promoter elements that bind to unique combinations of gap gene
proteins (reviewed in Akam, 1989). For example, the
protein products of the gap genes hunchback and
Kriippel bind to specific sites in the eve promoter
(Stanojevid et al. 1989), and deletion of these sites leads
12
S. A. Newman and W. D. Comper
to loss of specific stripes (Goto et al. 1989). The
implication of these studies is that each pair-rule stripe
is uniquely induced by a set of specific instructions, and
that dynamical pattern-generating systems are not
required for pattern formation (Akam, 1989).
If the possibility of an interplay between generic and
genetic mechanisms for the striped distribution of pairrule gene products is considered, they may be seen as
playing mutually reinforcing roles. One suggestion is
that feedback circuits may amplify or sharpen discontinuities and boundaries originally specified by genetic
'specific instruction' mechanisms (Akam, 1989).
Alternatively, the generic physical processes may actually be the primary ones (at least in evolutionary terms).
With the 'rough' pattern specified by a dynamical
reaction-diffusion mechanism, molecular evolution
would have had a globally organized morphological
substrate which it could stabilize and refine over time.
A second example of possible interactions between
genetic and reaction-diffusion mechanisms is provided
by the development of the vertebrate limb (Newman,
1988a) (Fig. 5G). Here the periodic pattern is generated sequentially, with the number of 'stripes' (corre-
sponding to parallel skeletal elements) increasing in a
discontinuous fashion over time. This pattern could be
generated by a mechanism like that schematized in
Fig. 6 (Newman and Frisch, 1979). The morphogen is
presumed to promote cellular aggregation or condensation, which precedes cartilage differentiation (Fell
and Canti, 1934; Thorogood and Hinchliffe, 1975). It
therefore provides a 'prepattern' for the skeleton. As is
typical for reaction-diffusion systems, the number of
morphogen peaks and valleys is sensitive to the size and
shape of the reaction vessel, or tissue domain. In this
model, the increase in peak number is tied to the
decrease in proximo-distal length of the undifferentiated zone, which appears to occur in relatively abrupt
steps (Summerbell, 1976). Each different-sized compartment will have a characteristic number of chemical
waves when the system reaches a steady state. (An
increase in anteroposterior length of this zone, as
occurs in the human limb, would also increase the peak
number in this model (Newman and Frisch, 1979)).
This dynamical pattern-forming mechanism may be
embodied in molecules such as TGF-/J (Massagud,
1987) which stimulates its own synthesis (Van Obber-
ao
o
o
Position in tissue
27
24
27-28
25
28
26
Fig. 5. Chemical wave
generation by a reactiondiffusion mechanism, and
examples of stripe patterns
during development.
(A-E) Graphical
representation of chemical
wave formation, based on
Maynard Smith (1968). See
text for discussion.
(F) Drosophila blastoderm
stage embryo showing early
even-skipped protein pattern
(white stripes). Picture
courtesy of R. Warrior and M.
Frasch. (G) Progress of
chondrogenesis in the chick
wing bud between 4 and 7 days
of development. Solid black
regions represent definitive
cartilage; stippled areas
represent early cartilage.
Stages are those of Hamburger
and Hamilton (1951). From
Newman and Frisch (1979),
copyright 1979 by the AAAS.
Physical mechanisms of morphogenesis and pattern formation
22'
St.
ghen-Schiller et al. 1988), and which also stimulates the
synthesis of the more slowly diffusing extracellular
matrix molecule fibronectin (Ignotz and Massagu6,
1986). Fibronectin, in turn, promotes precartilage cell
condensation (Frenz et al. 1989a,b). Lateral inhibition
of the positive feedback effects, which is required in
order for the condensations to be spatially confined
rather than encompassing the entire domain of competent tissue, may be the result of indirect effects.
While such reaction-diffusion mechanisms may regulate the development of a 'rough' skeletal pattern, they
cannot readily account for refinements such as the
anatomical distinctiveness of the radius and ulna, the
different digits, or the skeletons of the fore and hind
limbs as a whole. This probably requires the superimposition of 'genetic' modifiers such as homeobox-containing nuclear proteins (Gehring, 1987; Dolld et al. 1989a)
or retinoic acid receptors (Maden et al. 1988; D0II6 et al.
1989b). Homeobox proteins are nonuniformly distributed in developing limb buds (Oliver et al. 1988; Dolle"
et al. 1989t>) and may refine the rough pattern by
influencing local patterns of chondrogenic gene expression. Exogenously administered retinoids can
change the number of skeletal elements and affect the
symmetry of the pattern (Tickle et al. 1985). Endogenous retinoids (Thaller and Eichele, 1987) or their
receptors may modify the pattern directly, by their
effect on chondrogenesis, or indirectly, by their effect
13
Fig. 6. Interpretation of chick
limb development based on a
reaction-diffusion mechanism.
(A) (Left) Drawing of a 5-day
wing bud. (Right) Schematic
representation of 5-day wing
bud with as yet unpatterned
distal mesenchyme demarcated
by dashed line. (B) Graphs
representing predicted
distribution of TGF-/2 and
fibronectin in the
prechondrogenic distal
mesenchyme at approximate
stages 21 (top), 23 (middle),
and 25 (bottom) of
development. (C) Predicted
cartilage pattern based on
schematic model. A and B
adapted with changes from
Newman and Frisch (1979); C
from Newman and Frisch
(1979), copyright 1979 by the
AAAS.
on tissue growth (Ide and Aono, 1988; Paulsen and
Solursh, 1988).
Implications for evolution
The proposal that generic physical effects could be
responsible for major structural and patterning changes
during ontogeny immediately raises the question of the
significance of these processes in setting phylogenetic
trends. We have argued that machine-like molecular
mechanisms, in contrast to generic physical effects,
typically act locally rather than globally in spatial terms.
Analogously, the incremental events by which genetic
mechanisms evolve would most generally act 'locally' in
phylogenetic terms, conserving existing successful body
plans rather than causing species to undertake the first
steps leading to major structural rearrangements, the
adaptive significance of which would not generally be
realized in intermediate forms.
The action of generic physical mechanisms in morphogenesis and pattern formation provides a way out of
the conundrum of evolution through nonadaptive intermediates, for it suggests how, in systems embodying no
more than the standard nucleic-acid-based mechanisms
of inheritance, a genetically distant endpoint may be
immediately brought 'into sight'. We envision the
consequences of dominantly acting mutations that affect features of embryos and tissues susceptible to
14
S. A. Newman and W. D. Comper
generic physical mechanisms. As discussed above, these
mechanisms often exhibit nonlinear responses to
changes in control variables. Thus, a small change in
density of an ooplasmic determinant could lead to large
changes in its spatial distribution. A minor alteration in
interfacial tension between two tissue compartments
could strikingly change their relative configurations. A
pre-existing biosynthetic circuit which contains a diffusible component could be thrown into a pattern-generating mode by a small change in the ratio of reaction and
diffusion rates. In each of these cases, profound alterations in morphology, reproducible from generation to
generation, would ensue, virtually at one stroke. If the
resulting variants proved successful in establishing and
populating new niches, eons of genetic evolution could
follow, stabilizing and reinforcing the new outcome.
The alternative model, i.e. major morphological evolution by increments, would be analogous to bridging a
chasm of indeterminate breadth.
This scenario is consistent with the tempo of macroevolutionary change, which has been characterized as
consisting of long periods of morphological stasis punctuated by episodes of rapid structural reorganization
(Gould and Eldredge, 1978). It is also consistent with
the redundancy of morphogenetic mechanisms, and the
related 'overdetermination' of morphogenetic outcomes, that are well-recognized aspects of development. It should be noted that it has not been necessary
to invoke the concept of a 'genetic program', or the
change thereof, to account for ontogeny or phylogeny
in this perspective (see Oyama (1985), and Newman
(19886) for critical discussions of this concept). Neither
developmental 'routines' nor the evolutionary record
need be inscribed in the genes if the participation of
generic physical processes is taken into account.
Our perspective is complementary to the recognition
that static physical or 'architectural' constraints can
guide developmental outcomes (Horder and Martin,
1978; Springer and Mednick, 1985), or may give rise to
epiphenomena that can be coopted to new functions
during the course of evolution (Gould and Lewontin,
1979). But, by focusing on dynamical effects of a
generic nature, we suggest a role for these phenomena
beyond that of 'constraint'. In this interpretation,
genetic mechanisms would often serve to limit or
constrain pathways that have been set by generic
physical effects, a reversal of the usual attribution of all
morphological novelty to random genetic change.
Conclusions
We have described a number of effects that could lead
to the rearrangement of cytoplasmic and tissue components by virtue of these components having properties (such as viscoelasticity, surface tension and density
inhomogeneities) in common with ordinary non-living
materials. If excitable media (Ross et al. 1988) are
included among such materials, chemical waves can be
added to the list of patterning effects exhibited by both
nonliving and living systems.
Individual gene products, e.g. RNA and protein
molecules, are of course subject to generic chemical
effects, such as nucleophilic displacement (Cech, 1985)
and hydrophobic interactions (Chothia, 1984) in achieving their functional morphologies. We suggest that
increased size, chemical heterogeneity, and content of
macromolecules biosynthetically 'distant' from primary
gene products (e.g. glycosaminoglycans), have made
living systems increasingly subject to generic physical
effects. These in turn may have opened up new pathways for evolution (Comper, 1990), laying the basis for
tissue morphogenesis and pattern formation.
It is not only that generic physical processes might be
used during morphogenesis and pattern formation: in
many cases it would take special mechanisms to prevent
these effects from influencing biological outcomes.
Organisms may indeed incorporate examples of such
mechanisms: one function of the cytoskeleton may be
the prevention of nuclear sedimentation (Moroz, 1984).
However, it is reasonable to expect that generic
physicochemical effects would often be used, rather
than opposed, by living systems. It is also significant
that virtually all morphogenetic and patterning effects
seen in developing systems (e.g. epiboly, invagination,
involution, ingression, delamination, microfingering,
striping; see Gilbert, 1988) can, in principle, have
originally arisen by such generic effects.
The generic processes discussed above are capable of
interacting with one another in subtle and unexpected
ways. Convection in a multicomponent system can arise
from variations in interfacial tension, density or viscosity, individually or in concert, and in many cases
unravelling the contributions of these effects is technically difficult (Napolitano, 1984). As discussed above,
chemical potential differences can drive density inversions, leading to gravity-driven convection in systems
ostensibly in gravitational equilibrium (Comper et al.
19876). Moreover, reaction-diffusion systems with intrinsic pattern-forming capability are exquisitely sensitive to the presence of gravitational fields, which can
influence the pattern of chemical waves attained at
steady-state (Kondepudi and Prigogine, 1983). Because
of the chemical complexity of eggs, and of developing
systems in general, it would be expected that multiple
generic effects could contribute (along with locally
acting genetically specified molecular interactions) to
bringing about specific morphological outcomes.
Our overall viewpoint has some affinities to that
developed by D'Arcy Thompson in his book On
Growth and Form (1942, 1961). This author was also
concerned with the imprint of physical forces on biological form and pattern, and with distinctions (which
he acknowledged, were not always easy to make)
between 'properties of the organism...that are physical
in origin and those that are sui generis and peculiar to
living things' (D'Arcy Thompson, 1961; p. 13), a
dichotomy that roughly corresponds to our distinction
between generic and genetic processes. But D'Arcy
Thompson wrote virtually nothing about mechanisms of
development; his most compelling examples, such as
similarities in the arrangement of bone trabeculae to
Physical mechanisms of morphogenesis and pattern formation
the weight-bearing girders of a cantilever bridge, or the
possibility of deriving body or skull structure of one
species from another by coordinate transformation, are
rationalized on the basis of forces acting on adult forms.
They provide little insight into the mechanistic bases of
the ontogeny of these properties. The most important
deficiency in D'Arcy Thompson's analysis, however, is
his lack of serious consideration of mechanisms of
heredity, and in particular, of how generic physical
forces could act in a mutually reinforcing fashion with
genetic processes in giving form to organisms.
More recent discussions of the role of physical and
physicochemical processes in development (e.g. Meinhardt, 1982; Harrison, 1981), like that of D'Arcy
Thompson, have taken for granted the ability of hereditary mechanisms both to provide the necessary ingredients for, and to incorporate the effects of, these
processes. Mittenthal (1989) has gone beyond this by
explicitly considering a 'principle of matching' in which
genetically specified outcomes that meet certain physical constraints are most likely to be preserved by
evolution (this is similar to the 'Baldwin effect' (Simpson, 1953)). By assuming that generic effects are
efficacious developmental determinants at at least some
points in an organism's evolutionary history, our perspective provides a reasonable way for this matching to
come about.
Other general frameworks for morphogenesis and
pattern formation have deemphasized generic effects
while placing more of a burden on the efficacy of
presumed genetic programs. 'Positional information'
(Wolpert, 1969) is a model for pattern formation that
hypothesizes a genetically based potential of each cell in
an embryonic field to assume a biochemically unique,
site-specific state on the basis of simple cues provided
by monotonic gradients of signal molecules. The 'morphoregulator hypothesis' (Edelman, 1988) invokes a
hierarchy of rigidly programmed events in which the
establishment of tissue boundaries and organ form
depend exclusively on highly specific molecular interactions.
In contrast to genetic programming hypotheses,
which can potentially account for any imaginable form
or pattern, we have proposed an analysis of development that embodies generic mechanisms that act on
tissues and their components, either during the modern
ontogeny of an organism, or on its ontogeny sometime
in its evolutionary history. Changing patterns of gene
expression during development can drive morphogenesis and pattern formation by making tissues responsive
to fresh generic effects. Genetic change during evolution can act to conserve and reinforce these morphogenetic tendencies, or in rare instances, set phylogeny
on a new path by establishing susceptibility of the
embryo or its tissues to different generic forces. Such
generic-genetic interactions will not give rise to all
conceivable forms and patterns that may be constructed
from living cells and biological macromolecules. They
may nonetheless provide a concrete account of why
organisms achieve the particular variety of forms with
which we are so familiar.
15
We thank our colleagues who contributed to the original
research from our laboratories discussed in this paper. That
work has been supported by grants from the National Institutes of Health and National Science Foundation to S.A.N.,
and from the Australian Research Grant Scheme to W.D.C.
S.A.N. particularly acknowledges discussions with Drs G.
Forgacs, H.L. Frisch, N.S. Jaikaria, J. Mittenthal and D.
Noden. We also thank an anonymous referee for constructive
suggestions, and Mr Ryland Loos for preparing the line
drawings. This paper was written while S.A.N. was a Senior
International Fellow of the Fogarty International Center, in
residence at the Department of Biochemistry, Monash
University.
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